Morphologic and biochemical study of stimulated collagenogenesis

A study was made of the effect of potassium orotate on wound healing. The duration of wound healing was 3 days less in animals given potassium orotate as compared to controls, with the rate of prolin-containing protein synthesis by fibroblasts and the rate of their egress into the intercellular space being increased. No excess fibrillogenesis was noted. Hydroxyprolin content in the granulation tissue of wounds differed little in control and in experimental animals.     

Stimulation of fibrillogenesis in granulation tissue]

RNA synthesis by fibroblasts in healing wounds of mice and passage of the newly-formed RNA from the nucleus into the cytoplasm were studied in control mice and those treated by potassium orotate. Potassium orotate accelerated the passage of the RNA from the nucleus into the cytoplasm of the fibroblasts. Formation and maturation of the collagen fibrils in the wounds were studied by scanning electron microscope.     

Autoradiographic analysis of the effect of potassium orotate on the intensity of uridine-5 3H and proline-3H incorporation into wound fibroblasts]

The intensity of uridine-53H and proline-3H incorporation into fibroblast nuclei and cytoplasm as well as into intercellular spaces was studied in wound healing of mice given potassium orotate and in the control animals. It was shown that proline-3H incorporation was more intensive than that of uridine-53H. At the same time a significant increase in RNA synthesis in fibroblasts under the action of potassium orotate, and a less pronounced rise in proline-containing protein synthesis were noted in similar conditioelationships correlate with the fibroblast ultrastructural changes in the process of their differentiation.     

Electron-radioautographic study of the effect of potassium orotate on RNA and protein synthesis in fibroblasts during experimental wound healing

Electron microscopic radioautography with the use of the double-labeling technique was applied to study potassium orotate influence on RNA and protein synthesis in fibroblasts of intact mouse skin and in granulation tissue on days 3 and 7 of the healing of a musculocutaneous wound. Experimental animals received potassium orotate per os in a daily dose of 0.2 g/kg immediately after operation. Intact animals received the drug for 3 days. According to the assay data potassium orotate did not potentiate the protein-synthesizing activity of an individual cell but apparently activated the proliferation of wound fibroblasts.     

Objective Assessment of Endogenous Collagen In Vivo during Tissue Repair by Laser Induced Fluorescence

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm² He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications.     

Pyrimidine biosynthesis and its regulation in the estrogen-stimulated chick oviduct.

Studies on the incorporation of radio-labeled precursors into orotic acid and the pyrimidine nucleotides of RNA have established the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the chick oviduct. Measurements of the rate of incorporation of precursors into orotic acid in minces of oviduct revealed the activity of the orotate pathway to be accelerated in response to estrogen-stimulated nucleic acid synthesis and tissue growth. These data indicate that extrahepatic tissues of avian species meet their requirements for pyrimidine nucleotides through de novo synthesis rather than depend upon the liver or other exogenous sources for a supply of preformed pyrimidines. An examination of the influence of pyrimidine and purine nucleosides on the incorporation of radio-labeled precursors into orotic acid yielded evidence that pyrimidine biosynthesis in the chick is quite sensitive to inhibition by both purines and pyrimidines; the data indicate the reaction catalyzed by carbamoylphosphate synthetase to be the site of inhibition in both cases.     

Collagen-polysaccharide gel as a model of intercellular substance of the connective tissue]

Gel samples forming at 37 degrees C in the solutions containing tropocollagen and various polysaccharides were examined by electron microscopy. Contracting gel clots formed in the solutions containing chondroitin sulfate, proteoglycine from the tracheal cartilage, gum arabic. Electron microscopy showed such clots to be permeated with collagen fibrillae with transverse striations and a period of 640 A. An association between the density of the forming gel and the nature of the polysaccharide component is discussed. Gel forming in the solutions containing tropocollagen and various polysaccharides is regarded as a model of the connective tissue intercellular substance.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Peculiarities of nucleotide and nucleic acid metabolism in hydroxythiamine-induced vitamin B1 deficiency

24 hours after administration of hydroxythiamine, the vitamin B1 antimetabolite, the rat liver pools of orotic acid, UDP-glucose and ATP show a decrease. The cellular energy charge calculated from the adenylic nucleotide concentrations also appears to be significantly diminished. The de novo pyrimidine synthesis lowers under these conditions, while the rates of formation and destruction of essential UDP-sugars remain unaffected. The nucleic acid content is at the control level. A comparison of specific activities of UTP and RNA allows one to conclude that the previously observed decrease in [14C]orotate incorporation into RNA under the action of hydroxythiamine reflects the inhibition of RNA synthesis.     

The processing of procollagen in cultures of human and mouse fibroblasts.

Cultures of normal human and mouse fibroblasts convert procollagen to tropocollagen at varying rates. The conversion rate cannot be predicted from the species of origin of the fibroblast nor from the age of the donor tissue. Procollagen is converted to tropocollagen in both the extracellular space of the cell layer and in the culture medium. The collagen fibers of the cell layer are formed mostly from tropocollagen molecules generated in situ.     

SYNTHESIS, MIGRATION, AND RELEASE OF PRECURSOR COLLAGEN BY ODONTOBLASTS AS VISUALIZED BY RADIOAUTOGRAPHY AFTER [3H]PROLINE ADMINISTRATION

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30–40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280–350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.     

Effect of nifedipine and amlodipine on dead space wound healing in rats

Effect of two calcium channel blockers (CCBs) nifedipine and amlodipine, was studied on normal and steroid depressed wound healing in albino rats, using the dead space wound model. The drugs enhanced normal healing as evidenced by increase in tensile strength of 10 days old granulation tissue. There was neither a significant change in the hydroxyproline level (or collagen) nor a change in the glycosaminoglycan content in granulation tissue. However, lysyloxidase level was increased significantly. The increase in tensile strength could thus be attributed to better cross-linking and maturation of collagen rather than collagen synthesis per se. The drugs were also able to overcome steroid depressed wound healing. It is likely that the prohealing effects may be related to the improved antioxidant status too, since superoxide dismutase levels were observed to be higher in the CCB- treated animals.     

Type I collagen messenger RNA levels in experimental granulation tissue and silicosis in rats

A complementary DNA (cDNA) clone was constructed for chick pro alpha 2(I) collagen mRNA. This and previously constructed cDNA clones for chick and human pro alpha 1(I) collagen mRNAs were used to measure levels of type I procollagen messenger RNAs in two experimental models: viscose cellulose sponge-induced experimental granulation tissue and silica-induced experimental lung fibrosis in rats. Both Northern RNA blot and RNA dot hybridizations were used to quantitate procollagen mRNAs during formation of granulation tissue. The period of rapid collagen synthesis was characterized by high levels of procollagen mRNAs, which were reduced when collagen production returned to a low basal level. The rate of collagen synthesis and the levels of procollagen mRNAs during the period of rapid reduction in collagen production did not, however, parallel with each other. This suggests that translational control mechanisms are important during this time in preventing overproduction of collagen. In silicotic lungs, the early stages of fibroblast activation follow a similar path but appear faster. At a later stage, however, the RNA levels increase again and permit collagen synthesis to continue at a high rate, resulting in massive collagen accumulation.     

Amino acid metabolism of experimental granulation tissue in vitro

1. The intracellular volume in granulation tissue was about 15% of the total urea space. 2. The experimental granuloma has a greater ability to retain amino acids during the proliferation phase than later during the synthesis of collagen. 3. The synthesis of collagen and other proteins by granulation tissue is related to the concentrations of proline and glutamic acid in the medium. 4. The rate of synthesis of proline from glutamic acid in granulation-tissue slices is greatest during collagen synthesis. It is enhanced by lactate. 5. Extracellular cations influence the synthesis of collagen and ouabain is inhibitory. Synthesis of other proteins is less sensitive in this respect. 6. It is suggested that the synthesis of collagen is related to the supply of certain amino acids, especially proline, and hence to the redox balance, and also to the function of the cell wall.     

Effect of prednisolone on the intensity of nucleic acid synthesis by granulation tissue fibroblasts

An experimental study was made of the effect exerted by prednisolone in a dose of 4 mg/kg body weight on wound healing. It was shown that prednisolone reduces the period of inflammation, accelerates the formation of granulation tissue and epidermis. All these effects are accompanied by diminution of the mitotic activity of fibroblasts and dramatic increase in the intensity of RNA synthesis in them. Dislocation of newly synthetized RNA from the nucleus to the cytoplasm proceeds at a greater rate as compared to control. The differentiation of fibroblasts is accelerated.     

Effects of interleukin-8 on granulation tissue maturation

The inflammatory alpha-chemokine, interleukin-8 (IL-8), affects the function and recruitment of various inflammatory cells, fibroblasts, and keratinocytes. Gap junctions are anatomical channels that facilitate the direct passage of small molecules between cells. The hypothesis is that IL-8 enhances gap junctional intercellular communication (GJIC) between fibroblasts in granulation tissue, which increases the rate of granulation tissue maturation. In vitro, human dermal fibroblasts were incubated with IL-8 prior to scrape loading, a technique that quantifies GJIC. Polyvinyl alcohol (PVA) sponges were implanted within subcutaneous pockets in rats and received local injections of either IL-8 or saline and were harvested on day 11. In vitro, IL-8 treated fibroblasts demonstrated an increase in GJIC by scrape loading compared to saline treated controls. In vivo, IL-8 treated PVA sponges demonstrated a decrease in cell density and an increase in vascularization compared to saline controls by H&E staining. Polarized light viewed Sirius red-stained specimens demonstrated greater collagen birefringence intensity, indicating thicker, more-mature collagen fibers. IL-8 increases GJIC in cultured fibroblasts and induces a more rapid maturation of granulation tissue.     

Incorporation of exogenous precursors into uridine and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo

The possibility of compartmentation of UTP in vivo was investigated in the renal cortex of unanaesthetized rats. In addition, liver and spleen were studied in order to compare tissues with different utilization of precursors for pyrimidine nucleotide synthesis. After continuous 2h infusions of [(3)H]uridine or [(3)H]orotate, their incorporation into UTP, UDP-sugars and RNA was quantified. Rates of RNA synthesis were calculated by dividing the incorporation of precursor into RNA by the average specific radioactivity of the UTP pool. Although similar RNA-synthesis rates might have been expected with the two precursors, higher rates were found with uridine than with orotate. The relative incorporation into UDP-sugars of these precursors was also different. Similar results were obtained in the liver. In the spleen, equal amounts of both precursors were incorporated into UTP, but [(3)H]orotate incorporation did not lead to labelling of RNA. To evaluate the heterogeneity of cells with respect to the metabolism of pyrimidines, precursor incorporation was studied in isolated glomeruli and by radioautography. Incorporation into glomeruli was qualitatively similar to but quantitatively different from results in the renal cortex. Although there is obvious tissue heterogeneity, compartmentation of UTP pools is the most credible explanation for the results obtained with the renal cortex and liver. Consequently RNA and UDP-sugars may originate from two different UTP pools. Tissue heterogeneity is the likely explanation for the results obtained in the spleen. Studies of synthesis of pyrimidine and RNA, particularly in relation to growth and regeneration, must take into consideration the precursor used, the apparent existence of UTP compartmentation and the degree of cellular heterogeneity.     

The control of nucleic acid and nicotinamide nucleotide synthesis in regenerating rat liver

1. The effect of injecting nicotinamide on the incorporation of [(14)C]orotate into the hepatic nucleic acids of rats after partial hepatectomy was investigated. 2. At 3h after partial hepatectomy the rapid incorporation of [(14)C]orotate into RNA, and at 20h after partial hepatectomy the incorporation of [(14)C]orotate into both RNA and DNA, were inhibited in a dose-dependent fashion by the previous injection of nicotinamide. 3. The injection of nicotinamide at various times before the injection of [(14)C]orotate at 20h after partial hepatectomy revealed an inhibition of the incorporation of orotate into RNA and DNA which was non-linear with respect to the duration of nicotinamide pretreatment. 4. The induction of a hepatic ATP depletion by ethionine demonstrated that the synthesis of hepatic NAD and NADP in partially hepatectomized rats was more susceptible to an ATP deficiency than in control rats. 5. The total hepatic activity of ribose phosphate pyrophosphokinase (EC 2.7.6.1) was assayed at various times after partial hepatectomy and found to be only marginally greater than the maximum rate of hepatic NAD synthesis induced in vivo by nicotinamide injection between 12 and 24h after partial hepatectomy. 6. It is suggested that a competition exists between NAD synthesis and purine and pyrimidine nucleotide synthesis for available ATP and particularly 5-phosphoribosyl 1-pyrophosphate. In regenerating liver the competition is normally in favour of the synthesis of nucleic acid precursors, at the expense of NAD synthesis. This situation may be reversed by the injection of nicotinamide with a subsequent inhibition of nucleic acid synthesis.     

Histomorphological study of wound regeneration in animals irradiated for a long time with low-intensity microwaves]

Experimental study on 80 guinea pigs showed that in the animals exposed for a long time to the action of microwabes of low intensity the linear skin wounds healed by primary intension much more rapidly than in the non-irradiated animals. The postoperative scar was much more reliable in the irradiated animals. On the third day the irradiated animals were found to have a more intensive regeneration of the epithelium in the inflicted wound, and an intensive development of the granular tissue in the inferior portions. Later, an aceelerated synthesis of proteins, including that of collagen in the wound was observed; there was also a marked process of maturation of the granular tissue and formation of the intercellular substance. On the 7th-9th days fresh connective tissue (rich in fibroblasts with young fuchsinophilous collagen fibers) replaced the granular tissue; it was more mature than in the non-irradiated animals.